Methods for treating congestive heart failure

ABSTRACT

The invention features methods of treating or preventing congestive heart failure by administering a polypeptide containing an epidermal growth factor-like domain encoded by a neuregulin gene.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This work was supported in part by NIH Grants HL-38189, HL-36141, and aNASA award. The government has certain rights in the invention.

FIELD OF THE INVENTION

The field of the invention is treatment and prevention of congestiveheart failure.

BACKGROUND OF THE INVENTION

Congestive heart failure, one of the leading causes of death inindustrialized nations, results from an increased workload on the heartand a progressive decrease in its pumping ability. Initially, theincreased workload that results from high blood pressure or loss ofcontractile tissue induces compensatory cardiomyocyte hypertrophy andthickening of the left ventricular wall, thereby enhancing contractilityand maintaining cardiac function. However, over time, the leftventricular chamber dilates, systolic pump function deteriorates,cardiomyocytes undergo apoptotic cell death, and myocardial functionprogressively deteriorates.

Factors that underlie congestive heart failure include high bloodpressure, ischemic heart disease, exposure to cardiotoxic compounds suchas the anthracycline antibiotics, and genetic defects known to increasethe risk of heart failure.

Neuregulins (NRGs) and NRG receptors comprise a growth factor-receptortyrosine kinase system for cell-cell signalling that is involved inorganogenesis in nerve, muscle, epithelia, and other tissues (Lemke,Mol. Cell. Neurosci. 7: 247-262, 1996 and Burden et al., Neuron 18:847-855, 1997). The NRG family consists of three genes that encodenumerous ligands containing epidermal growth factor (EGF)-like,immunoglobulin (Ig), and other recognizable domains. At least 20(perhaps 50 or more) secreted and membrane-attached isoforms mayfunction as ligands in this signalling system. The receptors for NRGligands are all members of the EGF receptor (EGFR) family, and includeEGFR (or ErbB1), ErbB2, ErbB3, and ErbB4, also known as HER1 throughHER4, respectively, in humans (Meyer et al., Development 124: 3575-3586,1997; Orr-Urtreger et al., Proc. Natl. Acad. Sci. USA 90: 1867-71, 1993;Marchionni et al., Nature 362: 312-8, 1993; Chen et al., J. Comp.Neurol. 349: 389-400, 1994; Corfas et al., Neuron 14: 103-115, 1995;Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; andPinkas-Kramarski et al., Oncogene 15: 2803-2815, 1997).

The three NRG genes, Nrg-1, Nrg-2, and Nrg-3, map to distinctchromosomal loci (Pinkas-Kramarski et al., Proc. Natl. Acad. Sci. USA91: 9387-91, 1994; Carraway et al., Nature 387: 512-516, 1997; Chang etal., Nature 387: 509-511, 1997; and Zhang et al., Proc. Natl. Acad. Sci.USA 94: 9562-9567, 1997), and collectively encode a diverse array of NRGproteins. The most thoroughly studied to date are the gene products ofNrg-1, which comprise a group of approximately 15 distinctstructurally-related isoforms (Lemke, Mol. Cell. Neurosci. 7: 247-262,1996 and Peles and Yarden, BioEssays 15: 815-824, 1993). Thefirst-identified isoforms of NRG-1 included Neu Differentiation Factor(NDF; Peles et al., Cell 69, 205-216, 1992 and Wen et al., Cell 69,559-572, 1992), Heregulin (HRG; Holmes et al., Science 256: 1205-1210,1992), Acetylcholine Receptor Inducing Activity (ARIA; Falls et al.,Cell 72: 801-815, 1993), and the glial growth factors GGF1, GGF2, andGGF3 (Marchionni et al. Nature 362: 312-8, 1993).

The Nrg-2 gene was identified by homology cloning (Chang et al., Nature387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; andHigashiyama et al., J. Biochem. 122: 675-680, 1997) and through genomicapproaches (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). NRG-2cDNAs are also known as Neural- and Thymus-Derived Activator of ErbBKinases (NTAK; Genbank Accession No. AB005060), Divergent of Neuregulin(Don-1), and Cerebellum-Derived Growth Factor (CDGF; PCT application WO97/09425). Experimental evidence shows that cells expressing ErbB4 orthe ErbB2/ErbB4 combination are likely to show a particularly robustresponse to NRG-2 (Pinkas-Kramarski et al., Mol. Cell. Biol. 18:6090-6101, 1998). The Nrg-3 gene product (Zhang et al., supra) is alsoknown to bind and activate ErbB4 receptors (Hijazi et al., Int. J.Oncol. 13:1061-1067, 1998).

An EGF-like domain is present at the core of all forms of NRGs, and isrequired for binding and activating ErbB receptors. Deduced amino acidsequences of the EGF-like domains encoded in the three genes areapproximately 30-40% identical (pairwise comparisons). Further, thereappear to be at least two sub-forms of EGF-like domains in NRG-1 andNRG-2, which may confer different bioactivities and tissue-specificpotencies.

Cellular responses to NRGs are mediated through the NRG receptortyrosine kinases EGFR, ErbB2, ErbB3, and ErbB4 of the epidermal growthfactor receptor family. High-affinity binding of all NRGs is mediatedprincipally via either ErbB3 or ErbB4. Binding of NRG ligands leads todimerization with other ErbB subunits and transactivation byphosphorylation on specific tyrosine residues. In certain experimentalsettings, nearly all combinations of ErbB receptors appear to be capableof forming dimers in response to the binding of NRG-1 isoforms. However,it appears that ErbB2 is a preferred dimerization partner that may playan important role in stabilizing the ligand-receptor complex. Recentevidence has shown that expression of NRG-1, ErbB2, and ErbB4 isnecessary for trabeculation of the ventricular myocardium during mousedevelopment.

In view of the high prevalence of congestive heart failure in thegeneral population, it would be highly beneficial to prevent or minimizeprogression of this disease by inhibiting loss of cardiac function, andideally, by improving cardiac function for those who have or are at riskfor congestive heart failure.

SUMMARY OF THE INVENTION

We have found that neuregulins stimulate compensatory hypertrophicgrowth and inhibit apoptosis of myocardiocytes subjected tophysiological stress. Our observations indicate that neuregulintreatment will be useful for preventing, minimizing, or reversingcongestive heart disease resulting from underlying factors such ashypertension, ischemic heart disease, and cardiotoxicity.

The invention provides a method for treating or preventing congestiveheart failure in a mammal. The method involves administering apolypeptide that contains an epidermal growth factor-like (EGF-like)domain to the mammal, wherein the EGF-like domain is encoded by aneuregulin gene, and wherein administration of the polypeptide is in anamount effective to treat or prevent heart failure in the mammal.

In various preferred embodiments of the invention, the neuregulin genemay be the NRG-1 gene, the NRG-2 gene, or the NRG-3 gene. Furthermore,the polypeptide may be encoded by any of these three neuregulin genes.Still further, the polypeptide used in the method may be recombinanthuman GGF2.

In another preferred embodiment of the invention, the mammal is a human.

In other embodiments of the invention, the congestive heart failure mayresult from hypertension, ischemic heart disease, exposure to acardiotoxic compound (e.g., cocaine, alcohol, an anti-ErbB2 antibody oranti-HER2 antibody, such as HERCEPTIN®, or an anthracycline antibiotic,such as doxorubicin or daunomycin), myocarditis, thyroid disease, viralinfection, gingivitis, drug abuse; alcohol abuse, periocarditis,atherosclerosis, vascular disease, hypertrophic cardiomyopathy, acutemyocardial infarction or previous myocardial infarction, leftventricular systolic dysfunction, coronary bypass surgery, starvation,an eating disorder, or a genetic defect.

In another embodiment of the invention, an anti-ErB2 or anti-HER2antibody, such as HERCEPTIN®, is administered to the mammal eitherbefore, during, or after anthracycline administration.

In other embodiments of the invention, the polypeptide containing anEGF-like domain encoded by a neuregulin gene is administered before,during, or after exposure to a cardiotoxic compound. In yet otherembodiments, the polypeptide containing the EGF-like domain isadministered during two, or all three, of these periods.

In still other embodiments of the invention, the polypeptide isadministered either prior to or after the diagnosis of congestive heartfailure in the mammal.

In yet another embodiment of the invention, the polypeptide isadministered to a mammal that has undergone compensatory cardiachypertrophy.

In other preferred embodiments of the invention, administration of thepolypeptide maintains left ventricular hypertrophy, prevents progressionof myocardial thinning, or inhibits cardiomyocyte apoptosis.

In yet another embodiment of the invention, the polypeptide may beadministered by administering an expression vector encoding thepolypeptide to the mammal.

By “congestive heart failure” is meant impaired cardiac function thatrenders the heart unable to maintain the normal blood output at rest orwith exercise, or to maintain a normal cardiac output in the setting ofnormal cardiac filling pressure. A left ventricular ejection fraction ofabout 40% or less is indicative of congestive heart failure (by way ofcomparison, an ejection fraction of about 60% percent is normal).Patients in congestive heart failure display well-known clinicalsymptoms and signs, such as tachypnea, pleural effusions, fatigue atrest or with exercise, contractile dysfunction, and edema. Congestiveheart failure is readily diagnosed by well known methods (see, e.g.,“Consensus recommendations for the management of chronic heart failure.”Am. J. Cardiol., 83(2A):1A-38-A, 1999).

Relative severity and disease progression are assessed using well knownmethods, such as physical examination, echocardiography, radionuclideimaging, invasive hemodynamic monitoring, magnetic resonanceangiography, and exercise treadmill testing coupled with oxygen uptakestudies.

By “ischemic heart disease” is meant any disorder resulting from animbalance between the myocardial need for oxygen and the adequacy of theoxygen supply. Most cases of ischemic heart disease result fromnarrowing of the coronary arteries, as occurs in atherosclerosis orother vascular disorders.

By “myocardial infarction” is meant a process by which ischemic diseaseresults in a region of the myocardium being replaced by scar tissue.

By “cardiotoxic” is meant a compound that decreases heart function bydirecting or indirectly impairing or killing cardiomyocytes.

By “hypertension” is meant blood pressure that is considered by amedical professional (e.g., a physician or a nurse) to be higher thannormal and to carry an increased risk for developing congestive heartfailure.

By “treating” is meant that administration of a neuregulin orneuregulin-like polypeptide slows or inhibits the progression ofcongestive heart failure during the treatment, relative to the diseaseprogression that would occur in the absence of treatment, in astatistically significant manner. Well known indicia such as leftventricular ejection fraction, exercise performance, and other clinicaltests as enumerated above, as well as survival rates and hospitalizationrates may be used to assess disease progression. Whether or not atreatment slows or inhibits disease progression in a statisticallysignificant manner may be determined by methods that are well known inthe art (see, e.g., SOLVD Investigators, N. Engl. J. Med. 327:685-691,1992 and Cohn et al., N. Engl. J. Med. 339:1810-1816, 1998).

By “preventing” is meant minimizing or partially or completelyinhibiting the development of congestive heart failure in a mammal atrisk for developing congestive heart failure (as defined in “Consensusrecommendations for the management of chronic heart failure.” Am. J.Cardiol., 83(2A):1A-38-A, 1999). Determination of whether congestiveheart failure is minimized or prevented by administration of a neurgulinor neuregulin-like polypeptide is made by known methods, such as thosedescribed in SOLVD Investigators, supra, and Cohn et al., supra.

By “at risk for congestive heart failure” is meant an individual whosmokes, is obese (i.e., 20% or more over their ideal weight), has beenor will be exposed to a cardiotoxic compound (such as an anthracyclineantibiotic), or has (or had) high blood pressure, ischemic heartdisease, a myocardial infarct, a genetic defect known to increase therisk of heart failure, a family history of heart failure, myocardialhypertrophy, hypertrophic cardiomyopathy, left ventricular systolicdysfunction, coronary bypass surgery, vascular disease, atherosclerosis,alcoholism, periocarditis, a viral infection, gingivitis, or an eatingdisorder (e.g., anorexia nervosa or bulimia), or is an alcoholic orcocaine addict.

By “decreasing progression of myocardial thinning” is meant maintaininghypertrophy of ventricular cardiomyocytes such that the thickness of theventricular wall is maintained or increased.

By “inhibits myocardial apoptosis” is meant that neuregulin treatmentinhibits death of cardiomyocytes by at least 10%, more preferably by atleast 15%, still more preferably by at least 25%, even more preferablyby at least 50%, yet more preferably by at least 75%, and mostpreferably by at least 90%, compared to untreated cardiomyocytes.

By “neuregulin” or “NRG” is meant a polypeptide that is encoded by anNRG-1, NRG-2, or NRG-3 gene or nucleic acid (e.g., a cDNA), and binds toand activates ErbB2, ErbB3, or ErbB4 receptors, or combinations thereof.

By “neuregulin-1,” “NRG-1,” “heregulin,” “GGF2,” or “p185erbB2 ligand”is meant a polypeptide that binds to the ErbB2 receptor and is encodedby the p185erbB2 ligand gene described in U.S. Pat. No. 5,530,109; U.S.Pat. No. 5,716,930; and U.S. Ser. No. 08/461,097.

By “neuregulin-like polypeptide” is meant a polypeptide that possessesan EGF-like domain encoded by a neuregulin gene, and binds to andactivates ErbB-2, ErbB-3, ErbB-4, or a combination thereof.

By “epidermal growth factor-like domain” or “EGF-like domain” is meant apolypeptide motif encoded by the NRG-1, NRG-2, or NRG-3 gene that bindsto and activates ErbB2, ErbB3, ErbB4, or combinations thereof, and bearsa structural similarity to the EGF receptor-binding domain as disclosedin Holmes et al., Science 256:1205-1210, 1992; U.S. Pat. No. 5,530,109;U.S. Pat. No. 5,716,930; U.S. Ser. No. 08/461,097; Hijazi et al., Int.J. Oncol. 13:1061-1067, 1998; Chang et al., Nature 387:509-512, 1997;Carraway et al., Nature 387:512-516, 1997; Higashiyama et al., J.Biochem. 122: 675-680, 1997; and WO 97/09425).

By “anti-ErbB2 antibody” or “anti-HER2 antibody” is meant an antibodythat specifically binds to the extracellular domain of the ErbB2 (alsoknown as HER2 in humans) receptor and prevents the ErbB2(HER2)-dependent signal transduction initiated by neuregulin binding.

By “transformed cell” is meant a cell (or a descendent of a cell) intowhich a DNA molecule encoding a neuregulin or polypeptide having aneuregulin EGF-like domain has been introduced, by means of recombinantDNA techniques or known gene therapy techniques.

By “promoter” is meant a minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for cell type or physiological status (e.g.,hypoxic versus normoxic conditions), or inducible by external signals oragents; such elements may be located in the 5′ or 3′ or internal regionsof the native gene.

By “operably linked” is meant that a nucleic acid encoding a polypeptide(e.g., a cDNA) and one or more regulatory sequences are connected insuch a way as to permit gene expression when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences.

By “expression vector” is meant a genetically engineered plasmid orvirus, derived from, for example, a bacteriophage, adenovirus,retrovirus, poxvirus, herpesvirus, or artificial chromosome, that isused to transfer a polypeptide (e.g., a neuregulin) coding sequence,operably linked to a promoter, into a host cell, such that the encodedpeptide or polypeptide is expressed within the host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a representation of a semiquantitative RT-PCR analysisshowing expression of neuregulin receptors during cardiac developmentand in adult rat cardiomyocytes.

FIG. 1B is a representation of an assay showing tyrosine phosphorylationof the ErbB4 receptor in cardiomyocytes treated with recombinant humanglial growth factor 2 (rhGGF2).

FIGS. 2A and 2B are representations of photomicrographs showing stainingof neonatal rat ventricular myocytes for myosin heavy chain (FIG. 2A)and BrdU-positive nuclei (FIG. 2B).

FIG. 2C is a graph showing that rhGGF2 stimulates DNA synthesis(indicated by % BrdU-positive myocytes) in neonatal rat ventricularmyocytes.

FIGS. 3A and 3B are graphs showing that rhGGF2 stimulates DNA synthesis(indicated by % relative ³H-thymidine uptake) in neonatal ratventricular myocytes.

FIG. 4 is a graph showing that ErbB2 and ErbB4 mediate the effects ofGGF2 on relative ³H-thymidine uptake in neonatal rat ventricularmyocytes.

FIG. 5 is a graph showing that GGF2 promotes survival in primarycultures of neonatal rat ventricular myocytes.

FIGS. 6A-6C and 6E-6G are representations of photomicrographs showingthat GGF2 diminishes apoptotic cell death in primary cultures ofneonatal rat ventricular myocytes.

FIG. 6D is a graph showing that rhGGF2 diminishes apoptotic cell deathin primary cultures of neonatal rat ventricular myocytes (indicated by adecrease in the percentage of TUNEL-positive myocytes).

FIG. 6H is a graph showing that rhGGF2 diminishes apoptotic cell deathin primary cultures of neonatal rat ventricular myocytes (determined byflow cytometry analysis of the sub-G1 fraction following propidiumiodide staining of rhGGF2-treated cells).

FIGS. 7A and 7B are graphs showing that rhGGF2 increases survival anddecreases apoptotic cell death in primary cultures of adult ratventricular myocytes.

FIGS. 8A and 8B are representations of photomicrographs showing thatGGF2 induces hypertrophic growth of neonatal rat ventricular myocytes.

FIG. 8C is a representation of a Northern blot showing thatprepro-atrial natriuretic factor (prepro-ANF), a marker of ventricularhypertrophy, and α-skeletal actin are up-regulated in neonatal ratventricular myocytes treated with GGF2.

FIG. 8D is a graph showing that GGF2 stimulates protein synthesis(indicated by relative ³H-leucine uptake) in neonatal rat ventricularmyocytes.

FIGS. 9A-9C are photomicrographs showing that GGF2 induces hypertrophicgrowth in primary cultures of adult rat ventricular myocytes.

FIG. 9D is a representation of Northern blots showing that prepro-ANFand α-skeletal actin are up-regulated in adult rat ventricular myocytestreated with GGF2.

FIG. 9E is a graph showing that GGF2 stimulates protein synthesis(indicated by relative ³H-leucine uptake) in adult rat ventricularmyocytes.

FIGS. 10A and 10B are representations of ribonuclease protection assaysshowing expression levels of ErbB2 (FIG. 10A), ErbB4 (FIG. 10B), andβ-actin in the left ventricles of control and aortic stenosis rathearts.

FIG. 11 is a representation of a Northern blot showing expression of ANFand glyceraldehyde phosphate dehydrogenase (GAPDH, a housekeeping gene)in myocytes from left ventricles of control and aortic stenosis rathearts.

FIGS. 12A and 12B are representations of ribonuclease protection assaysshowing expression levels of ErbB2 (FIG. 12A), ErbB4 (FIG. 12B), andβ-actin in myocytes from the left ventricles of control and aorticstenosis rat hearts.

FIGS. 13A and 13B are representations of a Western blot showingexpression levels of ErbB2 in 6-week (FIG. 13A) and 22-week (FIG. 13B)aortic stenosis and control rat hearts.

FIGS. 13C and 13D are representations of a Western blot showingexpression levels of ErbB2 in 6-week (FIG. 13C) and 22-week (FIG. 13D)aortic stenosis and control rat hearts.

FIG. 14 is a graph showing that rat cardiomyocyte cultures pre-treatedwith IGF-1 or NRG-1 are less susceptible to daunorubicin-inducedapoptosis.

FIG. 15A is a representation of a phosphorylation assay showing thatIGF- and NRG-1-stimulated phosphorylation of Akt is inhibited by thePI-3 kinase inhibitor wortmannin.

FIG. 15B is a graph showing that IGF-1 and NRG-1 inhibition of caspase 3activation in cells exposed to daunorubicin is PI-3 kinase-dependent.

DETAILED DESCRIPTION OF THE INVENTION

We have found that neuregulins promote survival and hypertrophic growthof cultured cardiac myocytes through activation of ErbB2 and ErbB4receptors.

In addition, we have observed, in animals with experimentally-inducedintracardiac pressure overload, that cardiomyocyte ErbB2 and ErbB4levels are normal during early compensatory hypertrophy and decreaseduring the transition to early heart failure.

Together, our in vitro and in vivo findings show that neuregulins areinvolved in stimulating compensatory hypertrophic growth in response toincreased physiologic stress, as well as inhibiting apoptosis ofmyocardial cells subjected to such stress. These observations indicatethat neuregulin treatment will be useful for preventing, minimizing, orreversing congestive heart disease. While not wishing to be bound bytheory, it is likely that neuregulin treatment will strengthen thepumping ability of the heart by stimulating cardiomyocyte hypertrophy,and will partially or completely inhibit further deterioration of theheart by suppressing cardiomyocyte apoptosis.

Neuregulins

Polypeptides encoded by the NRG-1, NRG-2, and NRG-3 genes possessEGF-like domains that allow them to bind to and activate ErbB receptors.Holmes et al. (Science 256: 1205-1210, 1992) has shown that the EGF-likedomain alone is sufficient to bind and activate the p185erbB2 receptor.Accordingly, any polypeptide product encoded by the NRG-1, NRG-2, orNRG-3 gene, or any neuregulin-like polypeptide, e.g., a polypeptidehaving an EGF-like domain encoded by a neuregulin gene or cDNA (e.g., anEGF-like domain containing the NRG-1 peptide subdomains C-C/D or C-C/D′,as described in U.S. Pat. No. 5,530,109, U.S. Pat. No. 5,716,930, andU.S. Ser. No. 08/461,097; or an EGF-like domain as disclosed in WO97/09425) may be used in the methods of the invention to prevent ortreat congestive heart failure.

Risk Factors

Risk factors that increase the likelihood of an individual's developingcongestive heart failure are well known. These include, and are notlimited to, smoking, obesity, high blood pressure, ischemic heartdisease, vascular disease, coronary bypass surgery, myocardialinfarction, left ventricular systolic dysfunction, exposure tocardiotoxic compounds (alcohol, drugs such as cocaine, and anthracyclineantibiotics such as doxorubicin, and daunorubicin), viral infection,pericarditis, myocarditis, gingivitis, thyroid disease, genetic defectsknown to increase the risk of heart failure (such as those described inBachinski and Roberts, Cardiol. Clin. 16:603-610, 1998; Siu et al.,Circulation 8:1022-1026, 1999; and Arbustini et al., Heart 80:548-558,1998), starvation, eating disorders such as anorexia and bulimia, familyhistory of heart failure, and myocardial hypertrophy.

Accordingly, neuregulins may be administered to prevent or decrease therate of congestive heart disease progression in those identified asbeing at risk. For example, neuregulin administration to a patient inearly compensatory hypertrophy may permit maintenance of thehypertrophic state and may prevent the progression to heart failure. Inaddition, those identified to be at risk, as defined above, may be givencardioproctive neuregulin treatment prior to the development ofcompensatory hypertrophy.

Neuregulin administration to cancer patients prior to and duringanthracycline chemotherapy or anthracycline/anti-ErbB2 (anti-HER2)antibody (e.g., HERCEPTIN®) combination therapy may prevent thepatients' cardiomyocytes from undergoing apoptosis, thereby preservingcardiac function. Patients who have already suffered cardiomyocyte lossmay also derive benefit from neuregulin treatment, because the remainingmyocardial tissue will respond to neuregulin exposure by displayinghypertrophic growth and increased contractility.

Therapy

Neuregulins and polypeptides containing EGF-like domains encoded byneuregulin genes may be administered to patients or experimental animalswith a pharmaceutically-acceptable diluent, carrier, or excipient, inunit dosage form. Conventional pharmaceutical practice may be employedto provide suitable formulations or compositions to administer suchcompositions to patients or experimental animals. Although intravenousadministration is preferred, any appropriate route of administration maybe employed, for example, parenteral, subcutaneous, intramuscular,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, oral,or topical (e.g., by applying an adhesive patch carrying a formulationcapable of crossing the dermis and entering the bloodstream)administration. Therapeutic formulations may be in the form of liquidsolutions or suspensions; for oral administration, formulations may bein the form of tablets or capsules; and for intranasal formulations, inthe form of powders, nasal drops, or aerosols. Any of the aboveformulations may be a sustained-release formulation.

Methods well known in the art for making formulations are found in, forexample, “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Sustained-release,biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be used tocontrol the release of the compounds. Other potentially usefulparenteral delivery systems for administering molecules of the inventioninclude ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes. Formulations for inhalationmay contain excipients, for example, lactose, or may be aqueoussolutions containing, for example, polyoxyethylene-9-lauryl ether,glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

Gene Therapy

Neuregulins and neuregulin-like polypeptides containing neuregulinEGF-like domains may also be administered by somatic gene therapy.Expression vectors for neuregulin gene therapy (e.g., plasmids,artificial chromosomes, or viral vectors, such as those derived fromadenovirus, retrovirus, poxvirus, or herpesvirus) carry aneuregulin-encoding (or neuregulin-like polypeptide-encoding) DNA underthe transcriptional regulation of an appropriate promoter. The promotermay be any non-tissue-specific promoter known in the art (for example,an SV-40 or cytomegalovirus promoter). Alternatively, the promoter maybe a tissue-specific promoter, such as a striated muscle-specific, anatrial or ventricular cardiomyocyte-specific (e.g., as described inFranz et al., Cardiovasc. Res. 35:560-566, 1997), or an endothelialcell-specific promoter. The promoter may be an inducible promoter, suchas the ischemia-inducible promoter described in Prentice et al.(Cardiovasc. Res. 35:567-574, 1997). The promoter may also be anendogenous neuregulin promoter.

The expression vector may be administered as naked DNA mixed with orconjugated to an agent to enhance the entry of the DNA into cells, e.g.,a cationic lipid such as Lipofectin™, Lipofectamine™ (Gibco/BRL,Bethesda, Md.), DOTAP™ (Boeringer-Mannheim, Indianapolis, Ind.) oranalogous compounds, liposomes, or an antibody that targets the DNA to aparticular type of cell, e.g., a cardiomyocyte or an endothelial cell.The method of administration may be any of those described in theTherapy section above. In particular, DNA for somatic gene therapy hasbeen successfully delivered to the heart by intravenous injection,cardiac perfusion, and direct injection into the myocardium (e.g., seeLosordo et al., Circulation 98:2800-2804, 1998; Lin et al., Hypertension33:219-224, 1999; Labhasetwar et al., J. Pharm. Sci. 87:1347-1350, 1998;Yayama et al., Hypertension 31:1104-1110, 1998). The therapeutic DNA isadministered such that it enters the patient's cells and is expressed,and the vector-encoded therapeutic polypeptide binds to and activatescardiomyocyte ErbB receptors.

The following Examples will assist those skilled in the art to betterunderstand the invention and its principles and advantages. It isintended that these Examples be illustrative of the invention and notlimit the scope thereof.

Example I General Methods Preparation of Cardiac Myocyte and Non-MyocytePrimary Cultures

Neonatal rat ventricular myocyte (NRVM) primary cultures were preparedas described previously (Springhorn et al., J. Biol. Chem. 267:14360-14365, 1992). To selectively enrich for myocytes, dissociatedcells were centrifuged twice at 500 rpm for 5 min, pre-plated twice for75 min, and finally plated at low density (0.7−1×10⁴ cells/cm²) inDulbecco's modified Eagle's (DME) medium (Life Technologies Inc.,Gaithersburg, Md.) supplemented with 7% fetal bovine serum (FBS) (Sigma,St. Louis, Mo.). Cytosine arabinoside (AraC; 10 μM; Sigma) was added tocultures during the first 24-48 h to prevent proliferation ofnon-myocytes, with the exception of cultures used for thymidine uptakemeasurements. Unless otherwise stated, all experiments were performed36-48 h after changing to a serum-free medium, DME plus ITS (insulin,transferrin, and selenium; Sigma). Using this method, we routinelyobtained primary cultures with >95% myocytes, as assessed by microscopicobservation of spontaneous contraction and by immunofluorescencestaining with a monoclonal anti-cardiac myosin heavy chain antibody(anti-MHC; Biogenesis, Sandown, N.H.).

Primary cultures of cellular fractions isolated from neonatal heartsenriched in non-myocyte cells were prepared by twice passaging cellsthat adhered to the tissue culture dish during the preplating procedure.These non-myocyte cultures, which contained few anti-MHC-positive cells,were allowed to grow to subconfluence in DME supplemented with 20% FBSbefore switching to DME-ITS for a subsequent 36 to 48 h.

Isolation and preparation of adult rat ventricular myocyte (ARVM)primary cultures was carried out using techniques previously described(Berger et al., Am. J. Physiol. 266: H341-H349, 1994). Rod-shapedcardiac myocytes were plated in culture medium on laminin—(10 μg/ml;Collaborative Research, Bedford, Mass.) precoated dishes for 60 min,followed by one change of medium to remove loosely attached cells. Thecontamination of ARVM primary cultures by non-myocytes was determined bycounting with a haemocytometer and was typically less than 5%. All ARVMprimary cultures were maintained in a defined medium termed “ACCITT”(Ellingsen et al., Am. J. Physiol. 265: H747-H754, 1993) composed ofDME, supplemented with 2 mg/ml BSA, 2 mM L-carnitine, 5 mM creatine, 5mM taurine, 0.1 μM insulin, and 10 nM triiodothyronine with 100 IU/mlpenicillin and 100 μg/ml streptomycin. In experimental protocolsdesigned to examine myocyte survival and/or apoptosis, insulin wasomitted from the defined medium, which is therefore termed “ACCTT”.

PCR Analysis of ErbB Receptors in Rat Heart

cDNA sequences encoding portions of the C-termini of ErbB receptors wereamplified by using the following synthetic oligonucleotide primers:ErbB2A (5′-TGTGCTAGTCAAGAGTCCCAACCAC-3′: sense; SEQ ID NO: 1) and ErbB2B(5′-CCTTCTCTCGGTAC TAAGTATTCAG-3′: antisense; SEQ ID NO: 2) foramplification of ErbB2 codon positions 857 to 1207 (Bargmann et al.,Nature 319: 226-230, 1986); ErbB3A (5′-GCTTAAAGTGCTTGGCTCGGGTGTC-3′:sense; SEQ ID NO: 3) and ErbB3B (5′-TCCTACACACTGACACTTTCTCTT-3′:antisense; SEQ ID NO: 4) for amplification of ErbB3 codon positions 712to 1085 (Kraus et al., Proc. Natl. Acad. Sci. USA 86: 9193-9197, 1989);ErbB4A (5′-AATTCACCCATCAGAGTGACGTTTGG-3′: sense; SEQ ID NO: 5) andErbB4B (5′-TCCTGCAGGTAGTCTGGGTGCTG: antisense; SEQ ID NO: 6) foramplification of ErbB4 codon positions 896 to 1262 (Plowman et al.,Proc. Natl. Acad. Sci. USA 90: 1746-1750, 1993). RNA samples (1 μg) fromrat hearts or freshly isolated neonatal and adult rat ventricularmyocytes were reverse-transcribed to generate first-strand cDNA. The PCRreactions were performed in a final volume of 50 μl containingapproximately 50 ng of first-strand cDNAs for thirty cycles in aPTC-100™ Programmable Thermal Controller (MJ Research, Inc.; Watertown,Mass.). Each cycle included 30 sec at 94° C., 75 sec at 63° C., and 120sec at 72° C. Thirty μl aliquots of each reaction mixture were analyzedby electrophoresis in 1% agarose gels and by ethidium bromide staining.The PCR products were directly cloned into the TA cloning vector(Invitrogen Co., San Diego, Calif.) and verified by automatic DNAsequencing.

Analysis of ErbB Receptor Phosphorylation

To analyze which receptor subtypes were tyrosine-phosphorylated,neonatal and adult ventricular myocyte cells were maintained inserum-free medium for 24 to 48 h, and then treated with recombinanthuman glial growth factor 2 (rhGGF2) at 20 ng/ml for 5 min at 37° C.Cells were quickly rinsed twice with ice-cold phosphate-buffered saline(PBS) and lysed in cold lysis buffer containing 1% NP40, 50 mM Tris-HCl(pH 7.4), 150 mM NaCl, 1 mM ethylene glycol-bis(β-aminoethylether)-N,N,N′, N′-tetraacetic acid (EGTA), 1 mMethylenediaminetetraacetic acid (EDTA), 0.5% sodium deoxycholate, 0.1%SDS, 1 mM sodium orthovanadate, 10 mM sodium molybdate, 8.8 g/L sodiumpyrophosphate, 4 g/L NaFl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10μg/ml aprotinin, and 20 μM leupeptin. Lysates were centrifuged at12,000×g at 4° C. for 20 min, and aliquots of 500 μg (neonatal myocytes)or 2000 μg (adult myocytes) of supernatant were incubated with antibodyspecific to ErbB2 or ErbB4 (Santa Cruz Biotechnology Inc., Santa Cruz,Calif.) overnight at 4° C. and precipitated with protein A-agarose(Santa Cruz Biotechnology, Inc.). Immunoprecipitates were collected andreleased by boiling in sodium dodecyl sulfate (SDS) sample buffer.Samples were fractionated by SDS-polyacrylamide gel electrophoresis(SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes(Biorad Laboratories, Hercules, Calif.) and probed with a PY20antiphosphotyrosine antibody (Santa Cruz Biotechnology, Inc.). Fordetection of ErbB2, the supernatants were also immunoprecipitated with abiotinylated RC20 antiphosphotyrosine antibody (Upstate Biotechnology,Inc., Lake Placid, N.Y.) and blotted with a monoclonal antibody to ErbB2(Ab-2; Oncogene Research Products, Cambridge, Mass.).

Incorporation of [³H]Thymidine and [³H]Leucine

As an index of DNA synthesis, [³H]thymidine incorporation was measuredas described previously (Berk et al., Hypertension 13:305-314, 1989).After incubation for 36 to 48 h in serum-free medium (DME plus ITS), thecells were stimulated with different concentrations of rhGGF2 (CambridgeNeuroScience Co., Cambridge, Mass.) for 20 h. [³H]thymidine (0.7Ci/mmol; Dupont) was then added to the medium at a concentration of 5μCi/ml and the cells were cultured for another 8 h. Cells were washedwith PBS twice, 10% TCA once, and 10% TCA was added to precipitateprotein at 4° C. for 45 min. Parallel cultures of myocytes not exposedto rhGGF2 were harvested under the same conditions as controls. Theprecipitate was washed twice with 95% ethanol, resuspended in 0.15 NNaOH and saturated with 1 M HCl, then aliquots were counted in ascintillation counter. The results are expressed as relative cpm/dishnormalized to the mean cpm of control cells in each experiment. Forantibody blocking experiments, the same procedure was applied exceptthat the cells were preincubated with an antibody (0.5 g/ml) specificfor each neuregulin receptor (c-neu Ab-2, Oncogene Research Products;and ErbB3 or ErbB4, Santa Cruz Biotechnology), for 2 h prior to additionof either rhGGF2 or rhFGF2.

The rate of [³H]leucine uptake was used as an index of proteinsynthesis. For these experiments, 10 μM cytosine arabinoside was addedto the culture medium. Cells were grown in serum-free medium for 36 to48 h and then stimulated with different doses of rhGGF2. After 40 h,[³H]leucine (5 μCi/ml) was added for 8 h, and cells were washed with PBSand harvested with 10% TCA. TCA-precipitable radioactivity wasdetermined by scintillation counting as above.

5-Bromo-2′-Deoxy-Uridine Incorporation and Immunofluorescence Staining

Nuclear 5-bromo-2′-deoxy-uridine (BrdU) incorporation and a cardiacmuscle-specific antigen, myosin heavy chain (MHC), were simultaneouslyvisualized using double-indirect immunofluorescence. Primary NRVMcultures were maintained in DME plus ITS for 48 h and then stimulatedwith rhGGF2 (40 ng/ml) for 30 h. Control cultures were preparedsimilarly but without rhGGF2. BrdU (10 μM) was added for the last 24 h.Cells were fixed in a solution of 70% ethanol in 50 mM glycine buffer,pH 2.0, for 30 min at −20° C., rehydrated in PBS and incubated in 4 NHCl for 20 min. Cells were then neutralized with three washes in PBS,incubated with 1% FBS for 15 min, followed by a mouse monoclonalanti-MHC (1:300; Biogenesis, Sandown, N.H.) for 60 min at 37° C. Theprimary antibody was detected with TRITC-conjugated goat anti-mouse IgG(1:300, The Jackson Laboratory, Bar Harbor, Me.), and nuclear BrdUincorporation was detected with fluorescein-conjugated anti-BrdUantibody from an in situ cell proliferation kit (Boehringer Mannheim Co.Indianapolis, Ind.). The coverslips were mounted with Flu-mount (FisherScientific; Pittsburgh, Pa.), and examined by immunofluorescencemicroscopy. About 500 myocytes were counted in each coverslip and thepercentage of BrdU-positive myocytes was calculated.

For examination of changes in myocyte phenotype with rhGGF2, cells werefixed in 4% (w/v) paraformaldehyde for 30 min at room temperature,rinsed with PBS, permeabilized with 0.1% Triton X-100 for 15 min, andthen incubated with 1% FBS for another 15 min, followed by incubationwith anti-MHC (1:300) and visualized with TRITC-conjugated (NRVM) orFITC-conjugated (ARVM) second antibody. ARVM were examined using a MRC600 confocal microscope (BioRad; Hercules, Calif.) with a Kr/Ar laser.

Cell Survival Assay And Detection of Apoptosis

Cell viability was determined by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT,Sigma) cell respiration assay, which is dependent on mitochondrialactivity in living cells (Mosman, J. Immunol. Meth. 65:55-63, 1983).Primary cultures of NRVM after 2 days in serum-free medium werestimulated with different concentrations of rhGGF2 for either 4 or 6days. ARVM were maintained in ACCTT medium or ACCTT medium plusdifferent concentrations of rhGGF2 for 6 days. MTT was then incubatedwith the cells for 3 h at 37° C. Living cells transform the tetrazoliumring into dark blue formazan crystals that can be quantified by readingthe optical density at 570 nm after cell lysis with dimethylsulfoxide(DMSO; Sigma).

Apoptosis was detected in neonatal and adult myocytes using the terminaldeoxynucleotidyltransferase (TdT)-mediated dUTP nick end-labeling(TUNEL) assay. 3′-end labelling of DNA with fluorescein-conjugated dUTPwas done using an in situ cell death detection kit (Boehringer Mannheim,Indianapolis, Ind.) following the manufacturer's instructions. Cellswere counterstained with an anti-MHC antibody as described above, andthe nuclei were also stained with Hoescht 33258 (10 μM, Sigma) for 5min. More than 500 myocytes were counted in each coverslip and thepercentage of TUNEL positive myocytes was calculated.

Flow cytometric analysis of neonatal myocytes fixed in 70% ethanol/PBSand stained with propidium iodide was also performed to quantify thepercentage of cells undergoing apoptosis. This method is based upon theobservation that cells undergoing apoptosis have a hypo-diploid quantityof DNA and localize in a broad area below the G0/G1 peak on a DNAhistogram. Briefly, cells were collected by trypsinization, pooled withnonattached cells, and fixed in 70% ethanol. After being rinsed oncewith PBS, cells were incubated with a propidium iodide (20 μg/ml, Sigma)solution containing RNase A (5 Kunitz units/ml) at room temperature for30 min. Data were collected using a FACScan (Becton-Dickinson, San Jose,Calif.). For each sample, 10,000 events were collected. Aggregated cellsand extremely small cellular debris were gated out.

Isolation and Hybridization of RNA

Total cellular RNA was isolated by a modification of the acidguanidinium/thiocyanate phenol/chloroform extraction method (Chomczynskiand Sacchi, Anal. Biochem. 162:156-159, 1987) using the TRIZOL reagent(Life Technologies Inc., Gaithersburg, Md.). RNA was size-fractionatedby formaldehyde agarose gel electrophoresis, transferred to nylonfilters (Dupont, Boston, Mass.) by overnight capillary blotting andhybridized with cDNA probes labelled with [α-³²P]dCTP by random priming(Life Technologies Inc.). The filters were washed under stringentconditions and exposed to X-ray film (Kodak X-Omat AR, Rochester, N.Y.).Signal intensity was determined by densitometry (Ultrascan XL,Pharmacia). The following cDNA probes were used: rat prepro-atrialnatriuretic factor (prepro-ANF; a marker of cardiomyocyte hypertrophy)(0.6 kb of coding region) (Seidman, et al., Science 225:324-326, 1984),and rat skeletal α-actin (240 by of a 3′-untranslated region) (Shani etal., Nucleic Acids Res. 9:579-589, 1981). A ratglyceraldehyde-3-phosphate dehydrogenase (GAPDH; a housekeeping gene)cDNA probe (240 by of the coding region) (Tso et al., Nucleic Acids Res.13:2485-2502, 1985) was used as control for loading and transferefficiency.

Aortic Stenosis Model

Ascending aortic stenosis was performed in male Wistar weanling rats(body weight 50-70 g, 3-4 weeks, obtained from Charles River BreedingLaboratories, Wilmington, Mass.), as previously described (Schunkert etal., Circulation, 87:1328-1339, 1993; Weinberg et al. Circulation,90:1410-1422, 1994; Feldman et al., Circ. Res., 73:184-192, 1993;Schunkert et al., J. Clin. Invest. 96:2768-2774, 1995; Weinberg et al.,Circulation, 95:1592-1600, 1997; Litwin et al., Circulation,91:2642-2654; 1995). Sham-operated animals served as age-matchedcontrols. Aortic stenosis animals and age-matched sham-operated controlswere sacrificed after anesthesia with intraperitoneal pentobarbital 65mg/kg at 6 and 22 weeks after surgery (n=20-29 per group). Hemodynamicand echocardiographic studies in this model have shown that compensatoryhypertrophy with normal left ventricular (LV) cavity dimensions andcontractile indices is present 6 weeks after banding, whereas animalsdevelop early failure by 22 weeks after banding, which is characterizedby onset of LV cavity enlargement and mild depression of ejectionindices and pressure development per gram LV mass. In the present study,in vivo LV pressure measurements were performed prior to sacrifice aspreviously described (Schunkert et al., Circulation, 87:1328-1339, 1993;Weinberg et al. Circulation, 90:1410-1422, 1994; Feldman et al., Circ.Res., 73:184-192, 1993; Schunkert et al., J. Clin. Invest. 96:2768-2774,1995; Weinberg et al., Circulation, 95:1592-1600, 1997; Litwin et al.,Circulation, 91:2642-2654; 1995). The animals were also inspected forclinical markers of heart failure, including the presence of tachypnea,ascites, and pleural effusions. Both body weight and LV weight wererecorded.

LV Myocyte Isolation for RNA Extraction

In a subset of animals (n=10 per group), the heart was rapidly excisedand attached to an aortic cannula. Myocyte dissociation by collagenaseperfusion was performed as previously described (Kagaya et al.,Circulation, 94:2915-2922, 1996; Ito et al., J. Clin. Invest.99:125-135, 1997; Tajima et al., Circulation, 99:127-135, 1999). Toevalulate the percentage of myocytes in the final cell suspension,aliquots of myocytes were fixed, permeabilized and blocked. The cellsuspension was then incubated with antibodies against α-sarcomeric actin(mAb, Sigma, 1:20) and von Willebrand Factor (pAb, Sigma, 1:200) todistinguish between myocytes and endothelial cells. Secondary antibodies(goat anti-rabbit, goat anti-mouse pAb, Molecular probes, 1:400) with aTexas Red (or Oregon Green) conjugate were used as a detection system.Ninety-eight percent myocytes and less than 2% fragments of endothelialcells or unstained cells (fibroblasts) were routinely obtained.

RNA Analysis

Total RNA was isolated from control and hypertrophied myocytes (n=10hearts in each group), and from LV tissue (n=10 hearts in each group)using TRI Reagent (Sigma). Tissue and myocyte RNA were used for thefollowing protocols. Using myocyte RNA, Northern blots were used toassess message levels of atrial natriuretic peptide which werenormalized to GAPDH (Feldman et al., Circ. Res. 73:184-192, 1993; Tajimaet al., Circulation, 99:127-135, 1999). These experiments were done toconfirm the specificity of myocyte origin of the RNA using thismolecular marker of hypertrophy.

We also performed reverse transcription-polymerase chain reactions(RT-PCRs) for initial estimation of the presence of ErbB2, ErbB4 andneuregulin in samples derived from adult rat heart and adult myocytesusing the following pairs of primers: ErbB2 sense 5′ GCT GGC TCC GAT GTATTT GAT GGT 3′ (SEQ ID NO: 7), ErbB2 antisense 5′ GTT CTC TGC CGT AGGTGT CCC TTT 3′ (SEQ ID NO: 8) (Sarkar et al., Diagn. Mol. Pathol.2:210-218, 1993); ErbB3 sense 5′ GCT TAA AGT GCT TGG CTC GGG TGT C 3′(SEQ ID NO: 3), ErbB3 antisense 5′ TCC TAC ACA CTG ACA CTT TCT CTT 3′(SEQ ID NO: 4) (Kraus et al., Proc. Natl. Acad. Sci. USA 86:9193-9197;1989), ErbB4 sense 5′ AAT TCA CCC ATC AGA GTG ACG TTT GG 3′ (SEQ ID NO:5), ErbB4 antisense 5′ TCC TGC AGG TAG TCT GGG TGC TG 3′ (SEQ ID NO: 6)(Plowman et al., Proc. Natl. Acad. Sci. USA 90:1746-1750; 1993);neuregulin sense 5′ GCA TCA CTG GCT GAT TCT GGA G 3′ (SEQ ID NO: 9),neuregulin antisense 5′ CAC ATG CCG GTT ATG GTC AGC A 3′ (SEQ ID NO:10). The latter primers recognize nucleic acids encoded by the NRG-1gene, but do not discriminate between its isoforms. The amplificationwas initiated by 1 min of denaturation, 2 min of annealing at the genespecific temperature and 2 min extension at 72° C. The whole PCRreaction was electrophoresed on a 1% agarose gel and the PCR products ofexpected size were gel-purified.

After cloning these fragments into pGEM-T vector (Promega, Madison,Wis.), the correctness and orientation of those fragments within thevector was confirmed by sequencing. Cloned PCR fragments were used togenerate a radiolabeled riboprobe using the MAXIscript in vitrotranscription kit (Ambion, Inc., Austin, Tex.) and α-³²P-UTP. Theplasmids containing the ErbB2, ErbB4 or neuregulin fragment werelinearized and a radiolabeled probe was synthesized by in vitrotranscription with T7 or T3 RNA polymerase. The β-actin probe providedby the kit was transcribed with T7 or T3 polymerase and resulted in a330 and 300 bp fragment, respectively. 20 μg of total RNA was hybridizedto 5×10⁵ cpm of ErbB2, ErbB4 or neuregulin c-RNA together with 2×10⁴ cpmof β-actin for later normalization according to the RPA II kit (Ambion)protocol.

After digestion with RNase A/RNase T1, the samples were precipitated,dried, redissolved and finally separated on a 5% polyacrylamide gel for2 hours. The gel was exposed to Kodak MR film for 12-48 hours, and theassay was quantified by densitometric scanning of the auto-radiographusing Image Quant software (Molecular Dynamics, Inc., Sunnyvale,Calif.). ErbB2, ErbB4 and neuregulin mRNA levels were normalized toβ-actin.

Western Blotting of ErbB2 and ErbB4

LV tissue (n=5 hearts per group) was rapidly homogenized in a RIPAbuffer containing 50 mmol/L Tris HCl, pH 7.4, 1% NP-40, 0.1% SDS, 0.25%Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 Aprotinin, 1 μg/mlleupeptin, 1 μg/ml pepstatin and 1 mM Na₃PO4. Proteins were quantifiedusing the Lowry assay kit (Sigma). 50 μg of protein in Laemmli SDSsample buffer were boiled for 5 minutes and after centrifugation loadedonto a 10% SDS-PAGE gel. After electrophoresis, proteins weretransferred to a nitrocellulose membrane at 100 mA overnight. Thefilters were blocked with 0.05% Tween-20, 5% nonfat milk and thenincubated with anti-ErbB2 or anti-ErbB4 (Santa Cruz Biotechnology, eachdiluted 1:100, 1 μg/ml). After incubation with goat anti-rabbitperoxidase-conjugated secondary antibody diluted 1:2000, blots weresubjected to the enhanced chemiluminescent (ECL) detection method(Amersham, Life Science) and afterwards exposed to Kodak MR film for30-180 seconds. Protein levels were normalized to protein levels ofβ-actin detected with anti-β-actin (Sigma).

In Situ Hybridization for Neuregulin

10-μm cryostat sections of left ventricular tissue (n=2 control and6-week aortic stenosis hearts) were used for in situ hybridizations.Antisense and sense RNA probe was synthesized from cDNA fragments inpBluescript with either T7 or T3 RNA polymerase and digoxigenin-labeledUTP (DIG RNA Labeling Mix, Boehringer Mannheim). Tissue sections werefirst treated with 4% paraformaldehyde for 20 minutes, followed by 30minutes digestion with proteinase K (10 μg/ml) at 37° C. and another 5minutes of fixation in 4% paraformaldehyde.

Following the fixation, the slides were washed in PBS three times for 5minutes, after which the sections were immersed in 0.1 M triethanolaminechloride buffer with 0.25% acetic anhydride for 10 minutes to blockpolar and charged groups on the section and hence prevent nonspecificprobe binding. After washing the slides in 2×SSC, they were thenprehybridized (50% formamide, 2×SSC, 5% dextransulfate, 0.1% SDS,1×Denhardt's, 400 μg/ml denatured salmon sperm DNA) at 45° C. for 60minutes in a moist chamber charged with 50% formamide/2×SSC. After 1hour, the probes were added to the prehybridization solution and theslides were hybridized for 16-18 hours at 45° C.

Following overnight hybridization, slides were twice washed in 4×SSC for30 minutes at 45° C. while shaking, and then incubated with RNaseA (40μg/ml) in 500 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 8.0, for 30 minutes at37° C. to remove unhybridized probe. After RNase treatment, sectionswere immersed in 2×SSC at 50° C. for 30 minutes and then in 0.2×SSC atthe same temperature for another 30 minutes. The slides wereequilibrated with TBS I buffer (100 mM Tris, 150 mM NaCl, pH 7.5) andthen blocked with blocking reagent for 30 minutes at room temperatureaccording to the manufacturer's protocol (DIG Nucleic Acid DetectionKit, Boehringer Mannheim).

After removing the blocking reagent, the slides were immersed in TBS Ifor 1 minute and then the anti-DIG-AP conjugate solution (DIG NucleicAcid Detection Kit, Boehringer Mannheim) was applied to each section for1.5 hours at room temperature in a humid chamber. Afterwards, the slideswere washed in TBS I three times, 10 minutes per wash, to wash off theexcess antibody and equilibrated in TBS II (100 mM Tris, 100 mM NaCl, pH9.5, 50 mM MgCl₂.7H₂O) for 5 minutes. The color substrate was preparedaccording to the manufacturer's instructions and applied to each sectionuntil a blue-colored reaction became visible. The reaction was stoppedand the slides were washed in PBS and distilled water for 5 minuteseach. After a nuclear counter-staining the sections were dehydratedthrough an ethanol series, immersed in xylene and mounted bycover-slipping in Permount.

Statistical Analysis

All values are expressed as mean±SEM. Statistical analysis ofdifferences observed between the aortic stenosis groups (6 and 22 weeksafter banding) and the age-matched control groups was done by ANOVAcomparison. An unpaired Student's test was used for comparison among thegroups at the same age post-banding. Statistical significance wasaccepted at the level of p<0.05.

Example II Neuregulins Promote Survival and Growth of Cardiac Myocytes

Expression of Neuregulin Receptors in the Heart

To determine which of the NRG receptors (i.e., ErbB2, ErbB3, ErbB4) areexpressed in rat myocardium, RNA from rat heart tissues at successivestages of development, and from freshly isolated neonatal and adultventricular myocytes, were reverse-transcribed and amplified by PCR,using primers that flank the variable C-termini of ErbB receptors. FIG.1A shows the semiquantitative RT-PCR analysis of neuregulin receptormRNA levels during cardiac development. Total RNA from embryonic (E14,E16, and E19), neonatal (P1) or adult (Ad) rat heart, and from freshlyisolated neonatal rat ventricular myocytes (NRVM) or adult ratventricular myocytes (ARVM) was reverse-transcribed into cDNA andamplified with receptor isoform-specific primers (see Methods). GAPDHwas used as a control for reverse transcription, PCR amplification, andgel loading (“M” denotes 1 kb or 120 by DNA molecular weight standards).The RT-PCR products were verified by DNA sequencing.

All three ErbB receptors were expressed in the developing rat heart atmid-embryogenesis (E14), with the following rank order of their relativemRNA abundances: ErbB4>ErbB2>ErbB3. The expression of ErbB receptors wasdown-regulated later in embryogenesis. At E16 and E19, and at post-natalday 1 (P1), only ErbB2 and ErbB4 mRNAs could be detected. In adult ratheart, ErbB4 was still detectable, but its mRNA abundance was lower thanthat detected in embryonic and neonatal hearts, whereas ErbB2 mRNA and,rarely, ErbB3 mRNA could be detected only at low levels in adultmyocardium. In freshly isolated neonatal and adult rat ventricularmyocyte primary cultures, both ErbB2 and ErbB4 mRNA were readilydetectable by RT-PCR, although ErbB4 expression levels were consistentlyhigher than those of ErbB2. Furthermore, when using receptor-specificcDNA probes for ErbB2, ErbB3 and ErbB4, only transcripts for ErbB4 werereadily detectable in freshly isolated neonatal and adult ratventricular myocytes by Northern blot.

To determine which of the ErbB receptors were tyrosine-phosphorylatedfollowing neuregulin treatment, primary cultures of NRVM or ARVM,maintained in serum-free medium for 24 to 48 h, were treated either withor without neuregulin, i.e., recombinant human glial growth factor 2(rhGGF2) (20 ng/ml) for 5 min. ErbB4 receptor protein wasimmunoprecipitated with an anti-ErbB4 antibody from 500 μg of NRVMlysates or 2000 μg of ARVM lysates, and phosphorylated form of ErbB4 wasdetected by an anti-phosphotyrosine antibody. The blot shown in FIG. 1Bis representative of 3 independent experiments. As shown in FIG. 1B,phosphorylated ErbB4 is quite prominent in neonatal myocytes and lessrobust, but detectable, in adult myocytes, which is consistent with thelevels of ErbB4 mRNA abundance we observed above. Phosphorylated formsof ErbB2 and ErbB3 could not be detected even if immunoprecipitated withbiotinylated-antiphosphotyrosine antibody, consistent with themuch-reduced mRNA abundances for these two neuregulin receptors inpost-natal cardiac myocytes.

GGF2 Stimulates DNA Synthesis in Neonatal Rat Ventricular Myocytes

To investigate the ability of GGF2 to stimulate DNA synthesis in NRVMprimary cultures, myocytes maintained in serum-free medium for 2 dayswere subsequently treated with 40 ng/ml rhGGF2 for 30 h. DNA synthesiswas monitored by measuring the incorporation of either BrdU (FIG. 2B) or[³H]thymidine (FIGS. 3A and 3B), which were added to the media either 24h or 8 h, respectively, before termination of each experiment.

FIG. 2A shows myocyte myosin heavy chain in NRVM, visualized with aTRITC-conjugated goat anti-mouse antibody (red). FIG. 2B showsBrdU-positive nuclei visualized with a fluorescein-conjugated mouseanti-BrdU antibody (green). The scale bar for FIGS. 2A and 2B isequilvalent to 10 μm. FIG. 2C shows the percentage of BrdU-positivemyocytes under control conditions and in the presence of GGF2 (data aremean±SD for 3 experiments. *, p<0.01). As displayed in FIG. 2C, 40 ng/ml(approximately 0.7 nM) of rhGGF2 increased the percentage ofBrdU-labelled myocytes (from postnatal day 1 rat heart ventricles) byabout 80%, an increase in magnitude that was similar to that observedwith [³H]thymidine incorporation (FIG. 3A).

FIGS. 3A and 3B show the effects of GGF2 on DNA synthesis inmyocyte-enriched and non-myocyte fractions from rat ventricular myocyteprimary isolates. In FIG. 3A, NRVM-enriched primary isolates or a“non-myocyte”-enriched fraction (see Methods) were exposed to control(i.e., serum-free) medium alone (Ctl) or to medium containing either 40ng/ml rhGGF2 (GGF) or 7% fetal bovine serum (FBS). In FIG. 3B,concentration-dependent effect of GGF2 on NRVM DNA synthesis is shown.DNA synthesis was assessed by [³H]thymidine incorporation, and the dataare expressed as relative cpm/dish normalized to the mean cpm of controlcells in each experiment (mean±SD of triplicate analyses from threeindependent experiments; *, p<0.01 vs control). Twenty ng/ml of rhGGF2provoked an approximate 60% increase in [³H]thymidine incorporation intoNRVM, which was about half that observed with 7% FBS. The mitogeniceffect of rhGGF2 on NRVM was concentration-dependent, with about an 80%increase at 50 ng/ml (i.e., 0.9 nM) (FIG. 3B). GGF2 had similarmitogenic effects on BrdU or [³H]thymidine incorporation on ratembryonic ventricular myocytes (E19) and postnatal ventricular myocytes(P5), whereas concentrations of GGF2 as high as 100 ng/ml had no effecton DNA synthesis in adult rat ventricular myocyte primary cultures.

The effects of rhGGF2 on non-myocyte fractions obtained following thepreplating steps of the neonatal rat ventricular myocyte isolationprocedure also were investigated. As shown in FIG. 3A, rhGGF2 did notinduce any significant change in [3H]thymidine incorporation intonon-myocytes. This was in contrast to 7% FBS, which induced nearly a10-fold increase in [³H]thymidine incorporation into this cellpopulation. Therefore, GGF2 shows a relatively specific action oncardiac myocytes compared to a myocyte-depleted cell population which,using the method of myocyte isolation we employed here, is composedlargely of fibroblasts and endothelial cells.

To determine which of the known neuregulin receptors mediate themitogenic effect of GGF2 on fetal and neonatal ventricular myocytes, DNAsynthesis was measured in primary NRVM cultures after incubation withantibodies specific for ErbB2, ErbB3 and ErbB4. Neonatal myocytes werecultured for two days in serum-free medium, after which they weretreated for 30 h either without (control), or with rhGGF2 (10 ng/ml), orwith rhFGF2 (20 ng/ml), or with GGF2/FGF2 and antibodies to ErbB2, ErbB3or ErbB4, either alone or in combination as illustrated. Antibodies (0.5μg/ml/antibody) were preincubated with cells for 2 h before the additionof either GGF2 or FGF2. [³H]Thymidine was added during the last 8 h(data are expressed as relative cpm/dish normalized to the mean cpm ofcontrol cells in each experiment, and are presented as mean±SD; n=3independent experiments; *, p<0.04 vs rhGGF2 alone; #, p>0.1 vs rhGGF2alone).

As shown in FIG. 4, a monoclonal antibody against the extracellulardomain of c-neu/ErbB2, inhibited the GGF2-dependents increase in[³H]thymidine incorporation into NRVM by GGF2 could be inhibited.Similarly, an antibody directed against the C-terminus of ErbB4 alsoblocked about 50% of the increase in [³H]thymidine incorporation inducedby GGF2. A combination of these two antibodies had the same effect aseither the anti-ErbB2 or anti-ErbB4 antibodies alone. In contrast, anantibody to ErbB3 had no effect on GGF2-induced DNA synthesis. To verifythat the effects seen with the ErbB2 and ErbB4 antibodies were specificfor GGF, sister NRVM primary cultures were treated with 20 ng/ml rhFGF2(recombinant human bFGF). Neither antibody had any effect on theapproximately 2-fold increase in [³H]thymidine incorporation withrhFGF2. These results suggest that at least two of the known neuregulinreceptor tyrosine kinases were present and coupled to downstreamsignalling cascades in the neonatal ventricular myocyte.

GGF2 Promotes Cardiac Myocyte Survival In Vitro

During development, the net increase in the number of functionalembryonic myocytes is dependent on both myocyte proliferative capacityand survival. Therefore, it was of interest to determine whether GGF2could promote survival of cardiac myocytes in addition to proliferation.Primary cultures of NRVM maintained in serum-free medium, with orwithout 10 μM of cytosine arabinoside (AraC), were treated with theindicated concentrations of GGF2 for 4 days, and the relative numbers ofmetabolically active cells were determined by a MTT cell respirationassay (see Methods). Data are expressed as a percentage of the mean MTTactivity of myocytes in triplicate culture dishes on day 0 at the timeof the addition of GGF2. Data are shown as mean±SD (n=3 experiments; *,p<0.05 vs control). We observed that approximately 25% of cells die byday 4. In contrast, addition of GGF2 resulted in a 30% increase in MTTactivity compared to controls. The effect was concentration-dependentwith an EC50 of 0.2 ng/ml (FIG. 5). This survival effect was observed inNRVM primary cultures for up to 7 days; it was also observed in thepresence of cytosine arabinoside (AraC), an antiproliferative agent. Asshown in FIG. 5, the survival effect of GGF2 was observed at 4 days inthe continuous presence of cytosine arabinoside, with about 90% myocyteviability in the presence of 50 ng/ml rhGGF2 compared to approximately70% viability in control cultures. In contrast, GGF2 had no significanteffect on the survival of myocyte-depleted, “non-myocyte”-enrichedprimary isolates at 4 days.

We examined next whether the survival effect of GGF2 was mediated byinhibition of programmed cell death (apoptosis). Primary cultures ofNRVM 2 days in serum-free medium were maintained in either the absenceof rhGGF2 (FIG. 6A-6C) or in the presence of 20 ng/ml of rhGGF2 (FIG.6E-6G) for 4 days. Cells were then fixed and stained with anti-MHCantibody and a TRITC-conjugated secondary antibody to visualize myocytes(FIGS. 6A and 6E) or with fluorescein-conjugated dUTP (i.e., TUNEL) toreveal apoptotic cells (FIGS. 6B and 6F). The TUNEL-positive myocytesdisplayed cell shrinkage and chromatin condensation, which were alsoidentified by Hoescht 33258-staining (FIGS. 6C and 6G). Apoptosis wasquantified either by counting the number of TUNEL-positive myocytes(FIG. 6D) or by flow cytometry analysis of the sub-G1 fraction followingpropidium iodine-staining of primary NRVM cultures that had been treatedfor 4 days with the indicated concentrations of rhGGF2 (H). The datashown for FIG. 6D and FIG. 6H are given as mean±S.D for threeindependent experiments. The scale bar in FIGS. 6A-6C and 6E-6Grepresents 10 μM.

After 6 days in serum-free medium, about 17% of NRVM maintained undercontrol conditions at low density (i.e., subconfluent) exhibitedevidence of apoptosis as detected by TUNEL staining, with smallcondensed nuclei and cell shrinkage consistent with apoptotic cell death(FIGS. 6A-6C and 6E-6G). In the presence of 20 ng/ml rhGGF2, the numberof TUNEL positive myocytes declined to about 8% (FIG. 6D). The effect ofGGF2 on inhibiting apoptosis was also quantified using flow cytometricanalysis of propidium iodide-labelled NRVM primary cultures. After 4days in serum- and insulin-free medium, 22% of NRVM were hypodiploid,consistent with initiation of programmed cell death. In the presence ofrhGGF2 at concentrations above 10 ng/ml, less than 10% of NRVM exhibitedevidence of apoptosis (FIG. 6H).

The survival and antiapoptotic effects of GGF2 on the adult ratventricular myocyte (ARVM) were also examined by MTT cell respirationassay and TUNEL staining. In the experiment shown in FIG. 7A, primarycultures of ARVM were maintained in either a serum- and insulin-freemedium (i.e., “ACCTT”, see Methods), or ACCTT medium plus GGF2 for 6days. The number of metabolically active cells was determined by the MTTcell respiration assay, and the data are expressed as the relativeabsorbance normalized to the mean absorbance of untreated, controlcells. Each bar represents the mean±S.D (n=3 experiments; *, p<0.05 vscontrol). In the experiment shown in FIG. 7B, primary cultures of ARVMwere maintained in ACCTT medium (control) or ACCTT medium plus rhGGF2(25 ng/ml) for 3 days. After fixation with 4% paraformaldehyde, myocyteswere visualized with an anti-MHC antibody and a TRITC-conjugatedsecondary antibody, and apoptotic cells were identified by TUNELstaining. About 500 myocytes were counted on each coverslip (data aremean±S.D of three independent experiments; *, p<0.05 versus control).When compared to untreated ARVM primary cultures, in which more than 10%of cells were positive for TUNEL labelling, rhGGF2 (20 ng/ml)-treatedadult myocyte cultures exhibited only about 3% TUNEL-positive staining(FIG. 7B). These results indicate neuregulins act as survival factors atleast in part by preventing programmed cell death in both neonatal andadult ventricular myocytes.

GGF2 Induces Hypertrophic Growth of Cardiac Myocytes

In order to investigate whether neuregulin signalling can induce ahypertrophic (growth) response in cardiac myocytes, we examined theeffects of GGF2 on induction of myocyte hypertrophy in both neonatal andadult rat ventricular myocyte primary cultures. FIGS. 8A and 8B showphotomicrographs of subconfluent NRVM primary isolates incubated eitherwithout (FIG. 8A) or with (FIG. 8B) rhGGF2 (20 ng/ml) for 72 h inserum-free medium, after which cells were fixed and stained with anantibody to cardiac MHC (red, TRITC) and examined using indirectimmunofluorescence microscopy. The scale bar shown in the figurerepresents 10 μM. After a 72-hr incubation in serum-free medium with 20ng/ml (i.e., 0.36 nM) of rhGGF2, neonatal cardiac myocytes (NRVM)exhibited a significant increase in cell size and in myofibrillardevelopment.

A hypertrophic response in cardiac myocytes is characterized by a numberof phenotypic changes in addition to an increase in cell size, such asan increase in contractile protein content without cellularproliferation and the re-activation of an “embryonic” gene program.Therefore, we examined the effects of neuregulin on levels of prepro-ANFand skeletal α-actin mRNA (transcripts normally found in relatively lowabundance in neonatal and adult ventricular myocytes), and on[³H]leucine incorporation as an index of protein synthesis in NRVMprimary cultures. FIG. 8C shows a Northern blot analysis for prepro-ANFand skeletal α-actin mRNA from total RNA (20 μg/lane) from NRVMincubated either with or without rhGGF2 (20 ng/ml) for the timesindicated. Equal loading and transfer of RNA were confirmed by GAPDHhybridization. RhGGF2 (20 ng/ml) increased mRNA levels for prepro-ANFand skeletal-actin within 60 min, approximately doubling by 16 h.

To test the effect of GGF2 on protein synthesis, NVRM were cultured inserum-free medium for 24 h, after which they were treated with theindicated concentrations of rhGGF2 for 40 h, and pulsed with [³H]leucinefor 8 h before termination of GGF2 stimulation. The incorporation of[³H]leucine at each concentration of GGF2 was normalized to the proteincontent of each dish, and data are expressed as relative cpm/dishnormalized to the mean cpm of untreated control cells in each experiment(mean±S.D.; n=3 experiments; *, p<0.01 vs control). FIG. 8D shows thatGGF2 also stimulated [³H]leucine incorporation, with about a 120%increase at 48 h, at a concentration of 5 ng/ml. To minimize possibleconfounding effects of GGF2 on the rate of [³H]leucine uptake intonon-myocyte contaminant cells, these experiments were repeated in thecontinuous presence of cytosine arabinoside with similar results.

GGF2 also caused hypertrophic responses in cultured adult ratventricular myocytes (ARVM). Primary cultures of ARVM were plated oncoverslips in 24-well dishes and maintained for 5 days in ACCITT mediumeither without (FIG. 9A) or with rhGGF2 (20 ng/ml) (FIGS. 9B and 9C).Cells were fixed in 4% paraformaldehyde, stained with an antibody tomyosin heavy chain (green, FITC), and examined by confocal microscopy.The scale bars represent 10 μM. By 72 h in the continuous presence of 20ng/ml of rhGGF2, some adult myocytes had begun to develop“pseudopod”-like extensions, primarily from the region of theintercalated discs, and by 5 days, more than 60% of the GGF-treatedadult cardiomyocytes displayed phenotypic changes consistent with thoseillustrated in FIGS. 9B and 9C, whereas more than 80% of untreated ARVMmaintained the phenotype exhibited in FIG. 9A.

GGF2 also enhanced expression of prepro-ANF and skeletal α-actin inARVM. Primary isolates of ARVM were stimulated either with or without 20ng/ml rhGGF2 for the times indicated. Total RNA was isolated andanalyzed by Northern blot (25 μg/lane) using prepro-ANF and skeletalα-actin cDNA probes. Equal loading and transfer conditions wereconfirmed by GAPDH hybridization. Phenylephrine (PE, 10 μM) was used asa positive control for hypertrophic growth. As shown in FIG. 9D, rhGGF2(20 ng/ml) doubled prepro-ANF mRNA abundance in ARVM primary culturesafter 8 h, and this had increased 3- to 4-fold within 20 h. An increasein skeletal α-actin mRNA abundance was also observed that was greaterthan that seen with phenylephrine (10 μM), an α-adrenergic agonist knownto induce hypertrophic growth and reexpression of a number of fetalgenes in adult rat ventricular myocytes. Within 7 h, skeletal α-actinmRNA levels were easily detectable, and increased by an additional 250%by 30 h treatment with GGF2. Neither GGF2 nor phenylephrine had anyeffect on GAPDH mRNA abundance under the conditions employed here.

To test the effect of GGF2 on protein synthesis, ARVM (2 days in ACCITTmedium) were stimulated with increasing concentrations of rhGGF2 for 40h and [³H]leucine was added during the last 14 h. [³H]Leucine uptake inGGF2-treated cultures was normalized to the mean of [³H]leucine uptakein non-stimulated control myocytes. Data were also normalized to proteincontent of each dish to adjust for any variability in cell number amongdishes (mean±S.D; n=4; *, p<0.01 vs control). As illustrated in FIG. 9E,GGF2 induced a dose-dependent increase in [³H]leucine incorporation,with a 70% increase at a concentration of 5 ng/ml. Thus, this neuregulininduces phenotypic changes consistent with hypertrophic adaptation inboth neonatal and adult rat ventricular myocyte phenotypes atsubnanomolar concentrations.

Example III ErbB2 and ErbB4 Expression Levels Decrease in AorticStenosis Rats in Transition from Chronic Hypertrophy to Early HeartFailure LV Hypertrophy and Hemodynamics

As shown in Table 1, left ventricular (LV) weight and the LV/body weightratio were significantly (p<0.05) increased in the 6-week and 22-weekaortic stenosis animals compared with age-matched controls. The in vivoLV systolic pressure was significantly increased in both 6-week and22-week aortic stenosis animals compared with age-matched controls. Invivo LV end-diastolic pressure was also higher in aortic stenosisanimals compared to age-matched controls. Consistent with prior studiesin this model, LV systolic developed pressure per gram was significantlyhigher in 6-week aortic stenosis animals in comparison with age-matchedcontrols, but depressed in 22-week aortic stenosis animals. At 22-weekpost banding, the aortic stenosis animals also showed clinical markersof failure including tachypnea, small pleural and pericardial effusions.

TABLE 1 Left Ventricular Hypertrophy and Hemodynamics C (6 wks) LVH (6wks) C (22-wks) LVH (22 wks) BW (g) 397 ± 10  378 ± 15 590 ± 10  564 ±19 LV Wt (g) 1.25 ± 0.05  1.58 ± 0.06* 1.64 ± 0.07  2.46 ± 0.10* LVWt/BW (g/kg) 3.18 ± 0.13  4.40 ± 0.21* 2.84 ± 0.14  4.41 ± 0.20* LVEDP(mmHg) 4.8 ± 0.3  12.4 ± 0.7* 6.5 ± 0.8  15.7 ± 1.0* LVSP (mmHg) 104 ±3  181 ± 7* 129 ± 5  182 ± 9* LVdevP/g (mmHg/g) 84.2 ± 5.2  108.1 ± 6.8*82.4 ± 7.8   68.1 ± 4.2* Table 1 Legend: LVH, hearts with leftventricular hypertrophy, 6 and 22 weeks after aortic stenosis; C,age-matched controls; BW, body weight; LV Wt, left ventricular weight;LVEDP, LV end-diastolic pressure; LVSP, LV systolic pressure; LV devP,LV develop pressure per gram. Values are mean ± SEM; *p < 0.05 vs.age-matched controls; _p < 0.05 vs. 6-weeks LVH. n = 14-20 per group.

Expression of LV ErbB2, ErbB4 and Neuregulin in Aortic Stenosis

Using RT-PCR, we were able to detect ErbB2, ErbB4 and neuregulin mRNA,but not ErbB3 mRNA, in LV tissue derived from hearts of adult male ratswith and without left ventricular hypertrophy, as well as in normal andhypertrophied myocytes. FIG. 10A shows a ribonuclease protection assaydemonstrating LV ErbB2 and β-actin mRNA expression in 6-week aorticstenosis hearts and controls, and in 22-week aortic stenosis hearts andcontrols. FIG. 10B shows a ribonuclease protection assay demonstratingLV ErbB4 and β-actin mRNA expression in 6-week aortic stenosis heartsand controls, and 22-week aortic stenosis hearts and controls. Steadystate levels of ErbB2, ErbB4 and neuregulin mRNA levels in LV tissuefrom aortic stenosis rats and controls (n=5 hearts per group) were thenmeasured by ribonuclease protection assay (RPA) and normalized toβ-actin. The LV neuregulin mRNA levels were not significantly differentin tissue from 6-week aortic stenosis rats compared to age-matchedcontrols (0.68±0.12 vs. 0.45±0.12 units, NS) or 22-week aortic stenosisrats compared to age-matched controls (0.78±0.21 vs. 0.51±0.21 units,NS). Moreover, the LV ErbB2 and ErbB4 mRNA levels, which were normalizedto levels of β-actin, were preserved in 6-week aortic stenosis rats withcompensatory hypertrophy relative to controls. In contrast, LV ErbB2(p<0.05) and ErbB4 (p<0.01) message levels were significantly depressedin 22-week aortic stenosis rats at the stage of early failure (FIG. 10and Table 2).

TABLE 2 LV mRNA and Protein Levels of ErbB Receptors C (6 wks) LVH (6wks) C (22-wks) LVH (22 wks) mRNA (LV) ErbB2 0.354 ± 0.016 0.326 ± 0.0280.528 ± 0.072 0.301 ± 0.027* ErbB4 1.158 ± 0.036 1.088 ± 0.062 1.236 ±0.050 0.777 ± 0.082** mRNA (myocyte) ErbB2 0.755 ± 0.066 0.683 ± 0.0271.609 ± 0.089 0.493 ± 0.035** ErbB4 0.291 ± 0.024 0.266 ± 0.012 0.346 ±0.023 0.182 ± 0.014** protein (LV) ErbB2 1.228 ± 0.107 1.073 ± 0.0921.218 ± 0.198 0.638 ± 0.065* ErbB4 2.148 ± 0.180 1.968 ± 0.150 1.446 ±0.119 0.828 ± 0.068** Table 2 Legend: LVH, hearts with left ventricularhypertrophy, 6 and 22 weeks after aortic stenosis; C, age-matchedcontrols; left ventricular (LV) mRNA levels were measured byribonuclease protection assay and normalized to β-actin; mRNA levelswere measured in RNA from both LV tissue (mRNA, LV; n = 5 hearts pergroup) and from LV myocytes (mRNA, myocyte; ErbB2 n = 5 hearts pergroup; ErbB4 n = 3-4 hearts per group). LV protein levels were measuredin LV tissue (n = 5 per group) by Western blotting and normalized toβ-actin. Values are mean ± SEM; *p < 0.05 vs. age-matched controls; **p< 0.01 vs. age-matched controls.

We next examined gene expression in RNA from LV myocytes of 6-week and22-week aortic stenosis animals and controls. The specificity ofexpression in myocytes was determined by examining message levels ofatrial natriuretic peptide (ANP), a positive molecular marker ofpressure overload hypertrophy, using myocyte RNA and normalization tolevels of GAPDH. As shown in FIG. 11, ANP was upregulated in myocytesfrom both 6-week (710±16 vs. 230±40 units, p<0.05) and 22-weeks aorticstenosis animals (898±52 vs. 339±13 units, p<0.05) in comparison withcontrols (n=5 per group). Neuregulin was not detectable by RPA in RNAderived from myocytes in any group.

ErbB2 (n=5 per group) and ErbB4 (n=3-4 per group) message levels werealso measured in myocyte RNA from both aortic stenosis groups (FIG. 12and Table 2). FIG. 12A shows a ribonuclease protection assaydemonstrating LV myocyte ErbB2 and β-actin mRNA expression in 6-weeksaortic stenosis hearts and controls, and 22-weeks aortic stenosis heartsand controls. FIG. 12B shows a ribonuclease protection assaydemonstrating LV myocyte ErbB4 and β-actin mRNA expression in 6-weekaortic stenosis hearts and controls, and 22-week aortic stenosis heartsand controls. Consistent with the measurements in LV tissues samples,cardiomyocyte ErbB2 and ErbB4 mRNA levels, normalized to β-actin levels,are preserved relative to controls in 6-week aortic stenosis animals atthe stage of compensatory hypertrophy (NS). However, both ErbB2 andErbB4 expression are significantly downregulated in 22-week aorticstenosis animals at the transition to failure (p<0.01).

LV ErbB2 and ErbB4 Protein Levels

Western blotting using polyclonal antibodies for ErbB2 and ErbB4 wasperformed using protein samples derived from LV tissue of 6-week and22-week aortic stenosis rats in comparison with age-matched controls(n=5 per group). FIGS. 13A and 13B show Western blots showing LV ErbB2and β-actin protein levels in 6-week (FIG. 13A) aortic stenosis heartsand controls, and 22-week (FIG. 13B) aortic stenosis hearts andcontrols. FIGS. 13C and 13D show Western blots showing LV ErbB4 andβ-actin protein levels in 6-week (FIG. 13C) aortic stenosis hearts andcontrols, and 22-week (FIG. 13D) aortic stenosis hearts and controls.Densitometric signals of each receptor were normalized to signals ofβ-actin. As shown in FIGS. 13A-13D and Table 2, ErbB2 and ErbB4 mRNAexpression is preserved relative to controls in 6-weeks aortic stenosisanimals at the stage of compensatory hypertrophy (NS) but ErbB2 (p<0.05)and ErbB4 (p<0.01) are downregulated in 22-week aortic stenosis animalsduring early failure. Thus, a decrease in both LV message and proteinlevels of ErbB2 and ErbB4 is present at the stage of early failure inthis model of pressure overload.

In Situ Hybridization for Neuregulin

Antisense digoxigenin-labeled mRNA of neuregulin generated reproduciblehybridization signals on LV cryosections, whereas the correspondingsense transcript generated no signal above background. Neuregulinsignals in adult heart cryosections were observed in the endothelialcells of the cardiac microvasculature with minimal or no signal in othercell compartments. There was no difference between control and aorticstenosis animals.

Example IV Inhibition of Heart Failure in Aortic Stenosis Mice byPolypeptides that Contain a Neuregulin-1 EGF-Like Domain

The Examples above describe data showing that rhGGF2 suppressesapoptosis and stimulates cardiomyocyte hypertrophy in an ErbB2- andErbB4-dependent fashion. Moreover, ErbB2 and ErbB4 receptors aredown-regulated in the left ventricles of rats with pressureoverload-induced heart failure. Cardiomyocyte apoptosis is extremelyrare during the early compensatory hypertrophic stage in aortic stenosismice (i.e., 4 weeks after aortic banding), but consistently appearsduring the transition to early heart failure (i.e., 7 weeks after aorticbanding).

These above observations indicate that administration of polypeptidesthat have an EGF-like domain encoded by a neuregulin gene will be usefulin inhibiting the progression of and/or protecting against congestiveheart failure. While not wishing to be bound by theory, it is likelythat neuregulin treatment will strengthen the pumping ability of theheart by stimulating cardiomyocyte hypertrophy, and partially orcompletely prevent further deterioration of the heart by suppressingcardiomyocyte apoptosis.

One of ordinary skill in the art can readily determine the optimaldosage regimen required for providing prophylaxis against congestiveheart disease or for slowing or halting progression of already-existentheart disease, using one of the many animals models for congestive heartfailure that are known in the art. For example, as a starting point, therelative efficacy of a 0.3 mg/kg dose of GGF2 administered at earlystages and late stages of cardiac disease in the aortic stenosis mousemodel may be assessed as follows.

Group 1 (n=6); treated: injections of rhGGF2 (0.3 mg/kg given onalternate days), initiated 48 hours after aortic banding and continuedthrough week 7.

Group 2 (n=6); treated: injections of rhGGF2 (0.3 mg/kg given onalternate days), initiated at the beginning of week 4 after aorticbanding and continued through week 7.

Group 3 (n=6); control: sham injections, initiated 48 hours after aorticbanding and continued through week 7.

Group 4 (n=6); control: sham injections, initiated at the beginning ofweek 4 after aortic banding and continued through week 7.

Animals are sacrificed at the end of week 7. Prior to sacrifice, leftventricular hemodynamics are measured as described in Example I above,or using any standard protocol. Confocal microscopy may be used toquantitate myocyte growth (hypertrophy) and myocyte apoptosis by in situnick-end labeling (TUNEL) or similar techniques for measuring celldeath, using standard protocols or as described in Example I.

One of skill in the art will fully comprehend and know how to performthe experiments needed to determine the optimal neuregulin dosageregimen (e.g., amount of dose, frequency of administration, optimal timeduring the disease course to initiate neuregulin treatment) forminimizing, preventing, or even reversing congestive heart disease.

Example V NRG-1 Inhibits Anthracycline-Induced Apoptosis in Rat CardiacMyocytes

The anthracycline antibiotics (e.g., daunorubicin, and doxorubicin) havebeen a mainstay of cancer chemotherapy for more than 20 years. However,the short- and long-term cardiotoxicity of these drugs limits both theindividual dose and the cumulative dose that can be delivered to apatient.

There are two clinical types of anthracycline-induced cardiotoxicity.The acute type, which can occur after a single dose of anthracycline, ischaracterized by electrocardiographic changes, arrhythmias, and areversible decrease in ventricular contractile function. The chronic,delayed type is characterized by a largely irreversible decrease inventricular contractile function which progresses to dilatedcardiomyopathy and congestive heart failure. The incidence of thischronic cardiotoxicity is in direct proportion to the cumulativeanthracycline dose.

We have found that GGF2 (NRG-1) inhibits anthracycline-induced apoptosisin rat cardiac myocytes. FIG. 14 shows that rat cardiomyocyte culturespre-treated with IGF-1 or NRG-1 are less susceptible to apoptosis(indicated by TUNEL staining) induced by 1 μM daunorubicin. For IGF-1this protective effect is rapid, and can be achieved within 30 minutesof pre-incubation, similar to what was reported for fetal cardiacmyocytes. In contrast, this effect takes 24 hours of pre-incubation withNRG-1.

FIG. 15A shows that both IGF-1 and NRG-1 cause rapid phosphorylation ofAkt (FIG. 15A), and that this is inhibited by the PI-3 kinase inhibitorwortmannin. Akt has been implicated in mediating survival signals insome systems through phosphorylation and inactivation of thepro-apoptotic protein caspase 3. Either thirty minutes of pre-incubationwith IGF-1 or 24 hours of pre-incubation with NRG-1 preventanthracycline-induced activation of caspase 3. This effect, as well asthe survival effect of IGF-1, is completely prevented by wortmannin(FIG. 15B). Thus, activation of PI-3 kinase is necessary for thecytoprotective effect of IGF on myocytes. However, the lack ofcytoprotection by NRG-1 over the same time course indicates thatactivation of PI-3 kinase and Akt is not sufficient for cytoprotection.The relatively long NRG-1 exposure period needed for cytoprotectionsuggests that NRG-1-dependent protection of cardiomyocytes againstapoptosis requires de novo protein synthesis. Consistent with thisobservation, treatment of the cells with cyclohexamide inhibits theanti-apoptotic effect of NRG-1 on cardiomyocytes.

The results described above show that NRG-1 effectively inhibitsanthracycline-induced apoptosis. Therefore, NRG-1 could be used to limitor prevent cardiotoxicity in patients undergoing anthracyclinechemotherapy and to treat patients that have congestive heart failurecaused by cardiotoxicity induced by anthracyclines or other cardiotoxicagents.

Existing in the art are various well known animal models ofanthracycline-induced cardiotoxicity. Mouse, rat, rabbit, hamster, dog,swine, and monkey models for assessing the relative efficacy oftherapeutic compounds for ameliorating anthracycline-inducedcardiotoxicity are described in “Amelioration of Chemotherapy InducedCardiotoxicity” Semin. Oncol. 25(4)Suppl. 10, Aug. 1998 (see, e.g.,Myers, Semin. Oncol. 25:10-14, 1998; Herman and Ferrans, Semin. Oncol.25:15-21, 1998; and Imondi, Semin. Oncol. 25:22-30, 1998). These modelsmay be used to determine the optimal neuregulin or neuregulin-likepolypeptide treatment regimen (e.g., amount and frequency of dosage, andtiming relative to anthracycline administration), for minimizing,preventing, or reversing anthracycline-induced cardiotocicity.

Example VI Neuregulin-Dependent Inhibition of Cardiac Failure Induced byAnthracycline/Anti-ErbB2 (Anti-HER2) Combination Therapy

Various types of cancer cells display increased expression or increasedbiological activity of ErbB receptors. These transmembrane receptortyrosine kinases bind growth factors belonging to the neuregulin (alsoknown as heregulin) family. Expression of the ErbB2 receptor (also knownas HER2 and neu) in cancer cells has been correlated with increases inproliferation of carcinoma cells derived from various tissues,including, but not limited to, breast, ovary, prostate, colon, pancreas,and salivary gland.

Recently, it has been shown that HERCEPTIN® (Trastuzumab; Genentech,Inc., South San Francisco, Calif.), a humanized monoclonal antibody thatspecifically binds the extracellular domain of the human ErbB2 (HER2)receptor, inhibits the growth of breast carcinoma cells in vitro and invivo by down-regulating ErbB2 activity. A Phase III clinical trialevaluating the safety and efficacy of combining HERCEPTIN® therapy withtraditional anthracycline (doxorubicin) chemotherapy in breast cancerpatients showed that patients receiving the combination therapydisplayed greater tumor shrinkage and inhibition of cancer progressionthan patients receiving either therapy alone. However, patientsreceiving combination therapy also suffered increased cardiotoxicityrelative to patients receiving anthracycline therapy alone, indicatingthat anti-ErbB2 (anti-HER2) antibodies such as HERCEPTIN® increaseanthracycline-induced cardiotoxicity. In addition, patients that hadpreviously been treated with doxorubicin and later received HERCEPTIN®also showed an increased incidence of cardiotoxicity, relative topatients treated with doxorubicin alone.

Given the recently-shown success of HERCEPTIN®/anthracycline combinationtherapy in ameliorating ErbB2-overexpressing breast tumors, it is likelythat similar combination therapies will soon be used to treat otherErbB2-overexpressing tumors. However, the benefit/risk ratio ofanti-ErbB2 antibody/anthracycline combination therapy would be greatlyimproved if its associated cardiotoxicity could be decreased orprevented.

Animal models of anthracycline-induced cardiotoxicity (see, e.g., Hermanand Ferrans, Semin. Oncol. 25:15-21, 1998 and Herman et al. Cancer Res.58:195-197, 1998) are well-known in the art. Moreover, antibodies thatblock neuregulin binding to ErbB2 receptors, such as those describedabove, are well-known. By inducing anthracycline/anti-ErbB2antibody-dependent heart failure in known animal models foranthracycline toxicity, one of skill in the art will readily be able todetermine the neuregulin dosage regimen required to minimize or preventsuch heart failure.

Other Embodiments

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims.

1. A method for treating or preventing congestive heart failure in amammal, said method comprising administering a polypeptide comprising anepidermal growth factor-like (EGF-like) domain to said mammal, whereinsaid EGF-like domain is encoded by a neuregulin gene, wherein saidadministering is in an amount effective to treat or prevent heartfailure in said mammal. 2-6. (canceled)
 7. The method of claim 1,wherein said neuregulin gene is the NRG-3 gene.
 8. The method of claim7, wherein said polypeptide is encoded by the NRG-3 gene.
 9. The methodof claim 1, wherein said mammal is a human.
 10. The method of claim 1,wherein said congestive heart failure results from hypertension;ischemic heart disease; exposure to a cardiotoxic compound; myocarditis;thyroid disease; viral infection; gingivitis; drug abuse; alcohol abuse;periocarditis; atherosclerosis; vascular disease; hypertrophiccardiomyopathy; acute myocardial infarction; left ventricular systolicdysfunction; coronary bypass surgery; starvation; an eating disorder; ora genetic defect.
 11. The method of claim 10, wherein said mammal hasundergone a myocardial infarction.
 12. The method of claim 10, whereinsaid cardiotoxic compound is an anthracycline; alcohol; or cocaine. 13.The method of claim 12, wherein said anthracyline is doxorubicin, ordaunomycin.
 14. The method of claim 13, wherein an anti-ErbB2 oranti-HER2 antibody is administered to said mammal before, during, orafter anthracycline administration.
 15. The method of claim 10, whereinsaid cardiotoxic compound is an anti-ErbB2 or anti-HER2 antibody. 16.The method of claim 14 or 15, wherein said anti-ErbB2 or anti-HER2antibody is HERCEPTIN®.
 17. The method of claim 10, wherein saidpolypeptide is administered prior to exposure to said cardiotoxiccompound.
 18. The method of claim 10, wherein said polypeptide isadministered during exposure to said cardiotoxic compound.
 19. Themethod of claim 10, wherein said polypeptide is administered afterexposure to said cardiotoxic compound.
 20. The method of claim 1,wherein said polypeptide is administered prior to the diagnosis ofcongestive heart failure in said mammal.
 21. The method of claim 1,wherein said polypeptide is administered after the diagnosis ofcongestive heart failure in said mammal.
 22. The method of claim 1,wherein said polypeptide is administered to a mammal that has undergonecompensatory cardiac hypertrophy.
 23. The method of claim 1, whereinadministration of said polypeptide maintains left ventricularhypertrophy.
 24. The method of claim 1, wherein said method preventsprogression of myocardial thinning.
 25. The method of claim 1, whereinadministration of said polypeptide inhibits cardiomyocyte apoptosis. 26.The method of claim 1, wherein said polypeptide is administered byadministering an expression vector encoding said polypeptide to saidmammal.